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PURPOSE To evaluate the safety and efficacy of N-acetylmannosamine (ManNAc) in GNE myopathy, a genetic muscle disease caused by deficiency of the rate-limiting enzyme in N-acetylneuraminic acid (Neu5Ac) biosynthesis. METHODS We conducted an open-label, phase 2, single-center (NIH, USA) study to evaluate oral ManNAc in 12 patients with GNE myopathy (ClinicalTrials.gov NCT02346461). Primary endpoints were safety and biochemical efficacy as determined by change in plasma Neu5Ac and sarcolemmal sialylation. Clinical efficacy was evaluated using secondary outcome measures as part of study extensions, and a disease progression model (GNE-DPM) was tested as an efficacy analysis method. RESULTS Most drug-related adverse events were gastrointestinal, and there were no serious adverse events. Increased plasma Neu5Ac (+2,159 nmol/L, p

Objective Heart anomalies represent nearly one-third of all congenital anomalies. They are currently diagnosed using ultrasound. However, there is a strong need for a more accurate and less operator-dependent screening method. Here we report a metabolomics characterization of maternal serum in order to describe a metabolomic fingerprint representative of heart congenital anomalies. Methods Metabolomic profiles were obtained from serum of 350 mothers (280 controls and 70 cases). Nine classification models were built and optimized. An ensemble model was built based on the results from the individual models. Results The ensemble machine learning model correctly classified all cases and controls. Malonic, 3-hydroxybutyric and methyl glutaric acid, urea, androstenedione, fructose, tocopherol, leucine and putrescine were determined as the most relevant metabolites in class separation. Conclusion The metabolomic signature of second trimester maternal serum from pregnancies affected by a fetal heart anomaly is quantifiably different from that of a normal pregnancy. Maternal serum metabolomics is a promising tool for the accurate and sensitive screening of such congenital defects. Moreover, the revelation of the associated metabolites and their respective biochemical pathways allows a better understanding of the overall pathophysiology of affected pregnancies. This article is protected by copyright. All rights reserved.

Key Points Question Is alkaptonuria associated with thyroid dysfunction? Findings In this cohort study, 125 adults with alkaptonuria followed up for a median of 93 months were found to have a 16.0% prevalence of primary hypothyroidism. This prevalence was significantly higher than the 3.7% prevalence in the general population. Meaning Results of this study suggest that screening for hypothyroidism should be considered in patients with alkaptonuria., Importance Alkaptonuria is an autosomal recessive disorder caused by pathogenic variants in the HGD gene. Deficiency of the HGD enzyme leads to tissue deposition of homogentisic acid (HGA), causing severe osteoarthropathies and cardiac valve degeneration. Although HGD is vital for the catabolism of tyrosine, which provides the basis for thyroid hormone synthesis, the prevalence of thyroid dysfunction in alkaptonuria is unknown. Objective To assess thyroid structure and function in patients with alkaptonuria. Design, Setting, and Participants A single-center cohort study was conducted in a tertiary referral center including patients with alkaptonuria followed up for a median of 93 (interquartile range, 48-150) months between February 1, 2000, and December 31, 2018. The alkaptonuria diagnosis was based on clinical presentation and elevated urine HGA levels. A total of 130 patients were considered for participation. Main Outcomes and Measures Prevalence of thyroid dysfunction in adults with alkaptonuria compared with the general population. Thyrotropin and free thyroxine levels were measured by immunoassay and repeated in each patient a median of 3 (interquartile range, 2-22) times. Neck ultrasonographic scans were analyzed in a subset of participants. Logistic regression was used to test the association of thyroid dysfunction with age, sex, thyroid peroxidase (TPO) antibodies, serum tyrosine levels, and urine HGA levels. Results Of the 130 patients, 5 were excluded owing to thyroidectomy as the cause of hypothyroidism. The study cohort consisted of 125 patients; the median age was 45 (interquartile range, 35-51) years. Most of the patients were men (72 [57.6%]). The prevalence of primary hyperthyroidism was 0.8% (1 of 125 patients), similar to 0.5% observed in the general population (difference, 0.003; 95% CI, −0.001 to 0.04; P = .88). The prevalence of primary hypothyroidism was 16.0% (20 of 125 patients), which is significantly higher than 3.7% reported in the general population (difference, 0.12; 95% CI, 0.10-0.24; P, This cohort study examines thyroid function and structure in patients with alkaptonuria.

GNE myopathy is a rare, autosomal recessive, inborn error of sialic acid metabolism, caused by mutations in GNE, the gene encoding UDP-N-acetyl-glucosamine-2-epimerase/N-acetylmannosamine kinase. The disease manifests as an adult-onset myopathy characterized by progressive skeletal muscle weakness and atrophy. There is no medical therapy available for this debilitating disease. Hyposialylation of muscle glycoproteins likely contributes to the pathophysiology of this disease. N-acetyl-D-mannosamine (ManNAc), an uncharged monosaccharide and the first committed precursor in the sialic acid biosynthetic pathway, is a therapeutic candidate that prevents muscle weakness in the mouse model of GNE myopathy. We conducted a first-in-human, randomized, placebo-controlled, double-blind, single-ascending dose study to evaluate safety and pharmacokinetics of ManNAc in GNE myopathy subjects. Single doses of 3 and 6g of oral ManNAc were safe and well tolerated; 10g was associated with diarrhea likely due to unabsorbed ManNAc. Oral ManNAc was absorbed rapidly and exhibited a short half-life (~2.4h). Following administration of a single dose of ManNAc, there was a significant and sustained increase in plasma unconjugated free sialic acid (Neu5Ac) (Tmax of 8-11h). Neu5Ac levels remained above baseline 48h post-dose in subjects who received a dose of 6 or 10g. Given that Neu5Ac is known to have a short half-life, the prolonged elevation of Neu5Ac after a single dose of ManNAc suggests that intracellular biosynthesis of sialic acid was restored in subjects with GNE myopathy, including those homozygous for mutations in the kinase domain. Simulated plasma concentration-time profiles support a dosing regimen of 6g twice daily for future clinical trials.

Background GNE myopathy is a rare genetic disease characterized by progressive muscle atrophy and weakness. It is caused by biallelic mutations in the GNE gene that encodes for the bifunctional enzyme, uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase/N-acetylmannosamine (ManNAc) kinase. Typical characteristics of GNE myopathy include progressive myopathy, first involving anterior tibialis muscle and sparing the quadriceps, and rimmed vacuoles on muscle biopsy. Identifying biallelic mutations by sequencing of the GNE gene confirms the diagnosis of GNE myopathy. In a subset of patients, diagnostic confirmation is challenged by the identification of mutations in only one allele, suggesting mutations in deep intronic regions or regulatory regions. Methods We performed targeted sequencing and copy number variant (CNV) analysis of GNE in two siblings who clinically presented with GNE myopathy. Further molecular and biochemical studies were done to characterize the effect of a previously uncharacterized GNE mutation. Results We report two siblings of Indian descent with characteristic features of GNE myopathy, including progressive skeletal muscle weakness initially involving the anterior tibialis, and rimmed vacuoles on muscle biopsy, in which a heterozygous mutation, p.Val727Met, was identified in both affected siblings, but no other deleterious variants in either coding region or exon–intron boundaries of the gene. Subsequent insertion/deletion analysis identified a novel 11.3-kb deletion (Chr9 [GRCh37]: g.36257583_36268910del) encompassing the GNE promoter region, with breakpoints residing in Alu repeats. Gene expression analysis revealed reduced GNE mRNA and protein levels, confirming decreased expression of the deleted allele harboring the deletion. Conclusions We have identified GNE as one of the genes susceptible to Alu-mediated recombination. Our findings suggest that the deletion may encompass the promoter or another region necessary for GNE expression. In patients with typical manifestations of GNE myopathy and a single GNE variant identified, copy number variant (CNV) analysis may be useful in arriving at the diagnosis.

The biosynthesis of sialic acid (Neu5Ac) leads to the intracellular production of cytidine-5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac), the active sialic acid donor to nascent glycans (glycoproteins and glycolipids) in the Golgi. UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase myopathy is a rare autosomal recessive muscular disease characterized by progressive muscle weakness and atrophy. To quantify the intracellular levels of CMP-Neu5Ac as well as N-acetylmannosamine (ManNAc) and Neu5Ac in human leukocytes, we developed and validated robust liquid chromatography-tandem mass spectrometry methods. A fit-for-purpose approach was implemented for method validation. Hydrophilic interaction chromatography was used to retain three hydrophilic analytes. The human leukocyte pellets were lysed and extracted in a methanol-water mixture and the leukocyte extract was used for LC-MS/MS analysis. The lower limits of quantitation for ManNAc, Neu5Ac and CMP-Neu5Ac were 25.0, 25.0 and 10.0 ng/ml, respectively. These validated methods were applied to a clinical study.

Background: Alkaptonuria (AKU) is a rare autosomal recessive disorder caused by pathogenic variants in the gene encoding homogentisate-1,2-deoxygenase (HGD). Deficiency of HGD leads to tissue deposition of homogentisic acid (HGA), causing severe osteoarthropathies, aortic and mitral valve degeneration and kidney stones. HGD is vital for the catabolism of tyrosine, which provides the backbone of iodination into thyroxine (T4) within the thyroid gland. The goal of this study was to assess thyroid function (TFT) in patients (pts) with AKU. Methods: We performed a cohort study including pts with AKU followed at our center between 2000 and 2018. All pts had thyroid stimulation hormone (TSH), freeT4 (fT4), anti-thyroid peroxidase antibodies (anti-TPO-abs) measured repeatedly by immunoassay. In addition, a subset of patients underwent a thyroid ultrasound (US). Hypothyroidism (HYPO) was defined as TSH above the reference range (0.27-4.2 mIU/ml) with fT4 below the reference range (0.9-1.7 ng/dl) on two repeated tests or normalization of TSH on levothyroxine (LT4) replacement therapy. Subclinical HYPO (S-HYPO) was defined as TSH above the reference range with normal fT4 on two repeated tests. Results:The study cohort consisted of 130 pts with AKU, majority men (55.4%), aged 59.1±15 years and followed for 36 (range 1-63) months, during which median 10 (range 2-35) TFTs were performed. Twenty-four pts were diagnosed with HYPO, but 3 pts were excluded due to post-thyroidectomy etiology of HYPO, giving a prevalence of HYPO of 16.5% (21/127) - significantly higher than reported 5% of the general population (p=0.006). S-HYPO was diagnosed in 4.7% (6/127) and overt HYPO in 11.8% (15/127) pts, who were treated with a median LT4 dose of 100 mcg daily (1.4 mcg/kg). The median thyroid volume in HYPO pts was 7 cc (range 3.9-13). The prevalence of elevated anti-TPO-abs in the whole cohort was 14% (18/127), while its prevalence in HYPO group was 43% (9/21) - significantly lower than reported 89.8% for HYPO pts in the general population (p

We report a 25-year-old female confirmed to have Smith-Magenis syndrome (SMS) due to a de novo RAI1 variant. Her past history is significant for developmental and intellectual delay, early and escalating maladaptive behaviors, and features consistent with significant sleep disturbance, the etiology of which was not confirmed for over two decades. The diagnosis of SMS was initially suspected in 1998 (at age 12 years), but that was 5 years before the initial report of RAI1 variants as causative of the SMS phenotype; cytogenetic fluorescence in situ hybridization studies failed to confirm an interstitial deletion of 17p11.2. Re-evaluation for suspected SMS was pursued with RAI1 sequencing analysis in response to urgent parental concerns of escalating behaviors and aggression with subsequent incarceration of the subject for assault of a health professional. Genetic analysis revealed a de novo RAI1 (NM_030665.3) nonsense variant, c.5536C>T; p.Q1846X. This case illustrates the importance of confirming the SMS diagnosis, which is associated with cognitive and functional impairment, as well as significant psychiatric co-morbidities and behavioral problems. The diagnosis was particularly relevant to the legal discussion and determination of her competence to stand trial. As other similar cases may exist, this report will help to increase awareness of the possibility of a very late diagnosis of SMS, with the need for re-evaluation of individuals suspected to have SMS who were initially evaluated prior to the identification of the RAI1 gene. © 2016 Wiley Periodicals, Inc.

Over 50% of the causes of fetal malformations in humans are still unknown. Recent evidence suggests the relationship between environmental exposure to endocrine disruptors and fetal malformations. Our study aims to establish the role of Bisphenol A (BPA), if any, in altering human reproduction. We enrolled 151 pregnant women who were divided into two groups: case group (CS, n=101), women with established diagnosis of developmental defect, and control group (CL, n=50), pregnant women with normally developed fetus. Total, free and conjugated BPA were measured in their blood using GC-MS with isotopic dilution. The results show a correlation between environmental exposure to BPA and the genesis of fetal malformations. Conjugated BPA, which was higher in the CL, casts light on the hypothesis that a reduced ability to metabolize the chemical in the mother can concur to the occurrence of malformation. In a more detailed manner, in case of chromosomal malformations, the average value of free BPA appears to be nearly three times greater than that of the controls. Similarly, in case of central and peripheral nervous system non-chromosomal malformations, the value of free BPA is nearly two times greater than that of the controls.

Central nervous system anomalies represent a wide range of congenital birth defects, with an incidence of approximately 1% of all births. They are currently diagnosed using ultrasound evaluation. However, there is strong need for a more accurate and less operator-dependent screening method. To perform a characterization of maternal serum in order to build a metabolomic fingerprint resulting from congenital anomalies of the central nervous system. This is a case–control pilot study. Metabolomic profiles were obtained from serum of 168 mothers (98 controls and 70 cases), using gas chromatography coupled to mass spectrometry. Nine machine learning and classification models were built and optimized. An ensemble model was built based on results from the individual models. All samples were randomly divided into two groups. One was used as training set, the other one for diagnostic performance assessment. Ensemble machine learning model correctly classified all cases and controls. Propanoic, lactic, gluconic, benzoic, oxalic, 2-hydroxy-3-methylbutyric, acetic, lauric, myristic and stearic acid and myo-inositol and mannose were selected as the most relevant metabolites in class separation. The metabolomic signature of second trimester maternal serum from pregnancies affected by a fetal central nervous system anomaly is quantifiably different from that of a normal pregnancy. Maternal serum metabolomics is therefore a promising tool for the accurate and sensitive screening of such congenital defects. Moreover, the details of the most relevant metabolites and their respective biochemical pathways allow better understanding of the overall pathophysiology of affected pregnancies.

Hermansky-Pudlak Syndrome (HPS) is an autosomal-recessive condition characterized by oculocutaneous albinism and a bleeding diathesis due to absent platelet delta granules. HPS is a genetically heterogeneous disorder of intracellular vesicle biogenesis. We first screened all our patients with HPS-like symptoms for mutations in the genes responsible for HPS-1 through HPS-6 and found no functional mutations in 38 individuals. We then examined all eight genes encoding the biogenesis of lysosome-related organelles complex-1, or BLOC-1, proteins in these individuals. This identified a homozygous nonsense mutation in PLDN in a boy with characteristic features of HPS. PLDN is mutated in the HPS mouse model pallid and encodes the protein pallidin, which interacts with the early endosomal t-SNARE syntaxin-13. We could not detect any full-length pallidin in our patient's cells despite normal mRNA expression of the mutant transcript. We could detect an alternative transcript that would skip the exon that harbored the mutation, but we demonstrate that if this transcript is translated into protein, although it correctly localizes to early endosomes, it does not interact with syntaxin-13. In our patient's melanocytes, the melanogenic protein TYRP1 showed aberrant localization, an increase in plasma-membrane trafficking, and a failure to reach melanosomes, explaining the boy's severe albinism and establishing his diagnosis as HPS-9.

Background GNE myopathy is a rare genetic disease characterized by progressive muscle atrophy and weakness. It is caused by biallelic mutations in the GNE gene that encodes for the bifunctional enzyme, uridine diphosphate (UDP)‐N‐acetylglucosamine (GlcNAc) 2‐epimerase/N‐acetylmannosamine (ManNAc) kinase. Typical characteristics of GNE myopathy include progressive myopathy, first involving anterior tibialis muscle and sparing the quadriceps, and rimmed vacuoles on muscle biopsy. Identifying biallelic mutations by sequencing of the GNE gene confirms the diagnosis of GNE myopathy. In a subset of patients, diagnostic confirmation is challenged by the identification of mutations in only one allele, suggesting mutations in deep intronic regions or regulatory regions. Methods We performed targeted sequencing and copy number variant (CNV) analysis of GNE in two siblings who clinically presented with GNE myopathy. Further molecular and biochemical studies were done to characterize the effect of a previously uncharacterized GNE mutation. Results We report two siblings of Indian descent with characteristic features of GNE myopathy, including progressive skeletal muscle weakness initially involving the anterior tibialis, and rimmed vacuoles on muscle biopsy, in which a heterozygous mutation, p.Val727Met, was identified in both affected siblings, but no other deleterious variants in either coding region or exon–intron boundaries of the gene. Subsequent insertion/deletion analysis identified a novel 11.3‐kb deletion (Chr9 [GRCh37]: g.36257583_36268910del) encompassing the GNE promoter region, with breakpoints residing in Alu repeats. Gene expression analysis revealed reduced GNE mRNA and protein levels, confirming decreased expression of the deleted allele harboring the deletion. Conclusions We have identified GNE as one of the genes susceptible to Alu‐mediated recombination. Our findings suggest that the deletion may encompass the promoter or another region necessary for GNE expression. In patients with typical manifestations of GNE myopathy and a single GNE variant identified, copy number variant (CNV) analysis may be useful in arriving at the diagnosis.

Smith-Magenis syndrome (SMS), a neurodevelopmental disorder characterized by dysmorphic features, intellectual disability (ID), and sleep disturbances, results from a 17p11.2 microdeletion or a mutation in the RAI1 gene. We performed exome sequencing on 6 patients with SMS-like phenotypes but without chromosomal abnormalities or RAI1 variants. We identified pathogenic de novo variants in two cases, a nonsense variant in IQSEC2 and a missense variant in the SAND domain of DEAF1, and candidate de novo missense variants in an additional two cases. One candidate variant was located in an alpha helix of Necdin (NDN), phased to the paternally inherited allele. NDN is maternally imprinted within the 15q11.2 Prader-Willi Syndrome (PWS) region. This can help clarify NDN's role in the PWS phenotype. No definitive pathogenic gene variants were detected in the remaining SMS-like cases, but we report our findings for future comparison. This study provides information about the inheritance pattern and recurrence risk for patients with identified variants and demonstrates clinical and genetic overlap of neurodevelopmental disorders. Identification and characterization of ID-related genes that assist in development of common developmental pathways and/or gene-networks, may inform disease mechanism and treatment strategies.

GNE myopathy is a rare autosomal recessive muscle disease caused by mutations in GNE, the gene encoding the rate-limiting enzyme in sialic acid biosynthesis. GNE myopathy usually manifests in early adulthood with distal myopathy that progresses slowly and symmetrically, first involving distal muscles of the lower extremities, followed by proximal muscles with relative sparing of the quadriceps. Upper extremities are typically affected later in the disease. We report a patient with GNE myopathy who presented with asymmetric hand weakness. He had considerably decreased left grip strength, atrophy of the left anterior forearm and fibro-fatty tissue replacement of left forearm flexor muscles on T1-weighted magnetic resonance imaging. The patient was an endoscopist and thus the asymmetric hand involvement may be associated with left hand overuse in daily repetitive pinching and gripping movements, highlighting the possible impact of environmental factors on the progression of genetic muscle conditions.

GNE myopathy is an adult-onset progressive myopathy, resulting from mutations in GNE, the key enzyme of sialic acid synthesis. The pathomechanism of GNE myopathy likely involves aberrant sialylation, since administration of sialic acid itself, or its precursor, N-acetylmannosamine (ManNAc), rescued hyposialylation of GNE myopathy mice. Recently, clinical trials for GNE myopathy patients were initiated. A robust, noninvasive biomarker is highly desirable for diagnosis of GNE myopathy and for evaluating response to therapy. Since muscle biopsies of patients with GNE myopathy demonstrated hyposialylation of predominantly O-linked glycans, we analyzed the O-linked glycome of patients’ plasma proteins using mass spectrometry. Most patients showed increased plasma levels of the core 1 O-linked glycan, Thomsen-Friedenreich (T)-antigen and/or decreased amounts of its sialylated form, ST-antigen. In addition, compared to unaffected individuals, all analyzed patients had a consistently increased ratio of T-antigen to ST-antigen. Importantly, the T/ST ratios were in the normal range in a GNE myopathy patient treated with intravenous immunoglobulins as a source of sialic acid, indicating response to therapy. Natural history and clinical trial data will reveal whether T/ST ratios can be correlated to muscle function. These findings not only highlight plasma T/ST ratios as a robust blood-based biomarker for GNE myopathy, but may also help explain the pathology and course of the disease.

OPA3-related 3-methylglutaconic aciduria, or Costeff Optic Atrophy syndrome, is a neuro-ophthalmologic syndrome of early-onset bilateral optic atrophy and later-onset spasticity, and extrapyramidal dysfunction. Urinary excretion of 3-methylglutaconic acid and of 3-methylglutaric acid is markedly increased. OPA3-related 3-methylglutaconic aciduria is due to mutations in the OPA3 gene located at 19q13.2–13.3. Here we describe two siblings with novel compound heterozygous variants in OPA3: c.1A>G (p.1M>V) in the translation initiation codon in exon 1 and a second variant, c.142+5G>C in intron 1. On cDNA sequencing the c.1A>G appeared homozygous, indicating that the allele without the c.1A>G variant is degraded. This is likely due to an intronic variant; possibly the IVS1+5 splice site variant. The older female sibling initially presented with motor developmental delay and vertical nystagmus during her first year of life and was diagnosed subsequently with optic atrophy. Her brother presented with mildly increased hip muscle tone followed by vertical nystagmus within the first 6 months of life, and was found to have elevated urinary excretion of 3-methylglutaconic acid and 3-methylglutaric acid, and optic atrophy by 1.5 years of age. Currently, ages 16 and 7, both children exhibit ataxic gaits and dysarthric speech. Immunofluorescence studies on patient's cells showed fragmented mitochondrial morphology. Thus, though the exact function of OPA3 remains unknown, our experimental results and clinical summary provide evidence for the pathogenicity of the identified OPA3 variants and provide further evidence for a mitochondrial pathology in this disease.

Chromosomal anomalies (CA) are the most frequent fetal anomalies. To evaluate the diagnostic performance of a machine learning ensemble model based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester . This is a case-control pilot study. Metabolomic profiles have been obtained on serum of 328 mothers (220 controls and 108 cases), using gas chromatography coupled to mass spectrometry. Eight machines learning and classification models were built and optimized. An ensemble model was built using a voting scheme. All samples were randomly divided into two sets. One was used as training set, the other one for diagnostic performance assessment. Ensemble machine learning model correctly classified all cases and controls. The accuracy was the same for trisomy 21 and 18; also, the other CA were correctly detected. Elaidic, stearic, linolenic, myristic, benzoic, citric and glyceric acid, mannose, 2-hydroxy butyrate, phenylalanine, proline, alanine and 3-methyl histidine were selected as the most relevant metabolites in class separation. The proposed model, based on the maternal serum metabolomic fingerprint of fetal aneuploidies during the second trimester, correctly identifies all the cases of chromosomal abnormalities. Overall, this preliminary analysis appeared suggestive of a metabolic environment conductive to increased oxidative stress and a disturbance in the fetal central nervous system development. Maternal serum metabolomics can be a promising tool in the screening of chromosomal defects. Moreover, metabolomics allows to extend our knowledge about biochemical alterations caused by aneuploidies and responsible for the observed phenotypes.

The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) catalyzes the first two committed steps in sialic acid synthesis. Non-allosteric GNE gene mutations cause the muscular disorder GNE myopathy (also known as hereditary inclusion body myopathy), whose exact pathology remains unknown. Increased knowledge of GNE regulation, including isoform regulation, may help elucidate the pathology of GNE myopathy. While eight mRNA transcripts encoding human GNE isoforms are described, we only identified two mouse Gne mRNA transcripts, encoding mGne1 and mGne2, homologous to human hGNE1 and hGNE2. Orthologs of the other human isoforms were not identified in mice. mGne1 appeared as the ubiquitously expressed, major mouse isoform. The mGne2 encoding transcript is differentially expressed and may act as a tissue-specific regulator of sialylation. mGne2 expression appeared significantly increased the first two days of life, possibly reflecting the high sialic acid demand during this period. Tissues of the knock-in Gne p.M712T mouse model had similar mGne transcript expression levels among genotypes, indicating no effect of the mutation on mRNA expression. However, upon treatment of these mice with N-acetylmannosamine (ManNAc, a Gne substrate, sialic acid precursor, and proposed therapy for GNE myopathy), Gne transcript expression, in particular mGne2, increased significantly, likely resulting in increased Gne enzymatic activities. This dual effect of ManNAc supplementation (increased flux through the sialic acid pathway and increased Gne activity) needs to be considered when treating GNE myopathy patients with ManNAc. In addition, the existence and expression of GNE isoforms needs consideration when designing other therapeutic strategies for GNE myopathy.

We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky–Pudlak syndrome type 2. This was ruled out because of the presence of platelet δ-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient's melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient's G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual.

Hermansky-Pudlak Syndrome (HPS) is an autosomal-recessive condition characterized by oculocutaneous albinism and a bleeding diathesis due to absent platelet delta granules. HPS is a genetically heterogeneous disorder of intracellular vesicle biogenesis. We first screened all our patients with HPS-like symptoms for mutations in the genes responsible for HPS-1 through HPS-6 and found no functional mutations in 38 individuals. We then examined all eight genes encoding the biogenesis of lysosome-related organelles complex-1, or BLOC-1, proteins in these individuals. This identified a homozygous nonsense mutation in PLDN in a boy with characteristic features of HPS. PLDN is mutated in the HPS mouse model pallid and encodes the protein pallidin, which interacts with the early endosomal t-SNARE syntaxin-13. We could not detect any full-length pallidin in our patient's cells despite normal mRNA expression of the mutant transcript. We could detect an alternative transcript that would skip the exon that harbored the mutation, but we demonstrate that if this transcript is translated into protein, although it correctly localizes to early endosomes, it does not interact with syntaxin-13. In our patient's melanocytes, the melanogenic protein TYRP1 showed aberrant localization, an increase in plasma-membrane trafficking, and a failure to reach melanosomes, explaining the boy's severe albinism and establishing his diagnosis as HPS-9.

Griscelli syndrome (GS), a rare autosomal recessive disorder characterized by partial albinism and immunological impairment and/or severe neurological impairment, results from mutations in the MYO5A (GS1), RAB27A (GS2), or MLPH (GS3) genes. We identified a Hispanic patient born of a consanguineous union who presented with immunodeficiency, partial albinism, hepatic dysfunction, hemophagocytosis, neurological impairment, nystagmus, and silvery hair indicative of Griscelli syndrome type 2 (GS2). We screened for point mutations, but only exons 2–6 of the patient’s DNA could be PCR-amplified. Whole genome analysis using the Illumina® 1M-Duo DNA Analysis BeadChip identified a homozygous deletion in the patient’s DNA. The exact breakpoints of the 47.5-kb deletion were identified as chr15q15-q21.1: g.53332432_53379990del (NCBI Build 37.1); the patient lacks the promoter and 5’UTR regions of RAB27A, thus confirming the diagnosis of GS2.

Hereditary inclusion body myopathy (HIBM) is an autosomal recessive neuromuscular disorder associated with mutations in uridine diphosphate (UDP)-N-acetylglucosamine (GlcNAc) 2-epimerase (GNE)/N-acetylmannosamine (ManNAc) kinase (MNK), the bifunctional and rate-limiting enzyme of sialic acid biosynthesis. We developed individual GNE and MNK enzymatic assays and determined reduced activities in cultured fibroblasts of patients, with HIBM harboring missense mutations in either or both the GNE and MNK enzymatic domains. To assess the effects of individual mutations on enzyme activity, normal and mutated GNE/MNK enzymatic domains were synthesized in a cell-free in vitro transcription-translation system and subjected to the GNE and MNK enzymatic assays. This cell-free system was validated for both GNE and MNK activities, and it revealed that mutations in one enzymatic domain (in GNE, G135V, V216A, and R246W; in MNK, A631V, M712T) affected not only that domain's enzyme activity, but also the activity of the other domain. Moreover, studies of the residual enzyme activity associated with specific mutations revealed a discrepancy between the fibroblasts and the cell-free systems. Fibroblasts exhibited higher residual activities of both GNE and MNK than the cell-free system. These findings add complexity to the tightly regulated system of sialic acid biosynthesis. This cell-free approach can be applied to other glycosylation pathway enzymes that are difficult to evaluate in whole cells because their substrate specificities overlap with those of ancillary enzymes.

The GNE gene encodes the rate-limiting, bifunctional enzyme of sialic acid biosynthesis, uridine diphosphate-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE). Biallelic GNE mutations underlie GNE myopathy, an adult-onset progressive myopathy. GNE myopathy-associated GNE mutations are predominantly missense, resulting in reduced, but not absent, GNE enzyme activities. The exact pathomechanism of GNE myopathy remains unknown, but likely involves aberrant (muscle) sialylation. Here, we summarize 154 reported and novel GNE variants associated with GNE myopathy, including 122 missense, 11 nonsense, 14 insertion/deletions, and seven intronic variants. All variants were deposited in the online GNE variation database (http://www.dmd.nl/nmdb2/home.php?select_db=GNE). We report the predicted effects on protein function of all variants well as the predicted effects on epimerase and/or kinase enzymatic activities of selected variants. By analyzing exome sequence databases, we identified three frequently occurring, unreported GNE missense variants/polymorphisms, important for future sequence interpretations. Based on allele frequencies, we estimate the world-wide prevalence of GNE myopathy to be ∼4-21/1,000,000. This previously unrecognized high prevalence confirms suspicions that many patients may escape diagnosis. Awareness among physicians for GNE myopathy is essential for the identification of new patients, which is required for better understanding of the disorder's pathomechanism and for the success of ongoing treatment trials.

Summary Background and objectives Elevated serum vitamin D with hypercalciuria can result in nephrocalcinosis and nephrolithiasis. This study evaluated the cause of excess 1,25-dihydroxycholecalciferol (1α,25(OH)2D3) in the development of those disorders in two individuals. Design, setting, participants, & measurements Two patients with elevated vitamin D levels and nephrocalcinosis or nephrolithiasis were investigated at the National Institutes of Health (NIH) Clinical Center and the NIH Undiagnosed Diseases Program, by measuring calcium, phosphate, and vitamin D metabolites, and by performing CYP24A1 mutation analysis. Results Both patients exhibited hypercalciuria, hypercalcemia, low parathyroid hormone, elevated vitamin D (1α,25(OH)2D3), normal 25-OHD3, decreased 24,25(OH)2D, and undetectable activity of 1,25(OH)2D-24-hydroxylase (CYP24A1), the enzyme that inactivates 1α,25(OH)2D3. Both patients had bi-allelic mutations in CYP24A1 leading to loss of function of this enzyme. On the basis of dbSNP data, the frequency of predicted deleterious bi-allelic CYP24A1 variants in the general population is estimated to be as high as 4%–20%. Conclusions The results of this study show that 1,25(OH)2D-24-hydroxylase deficiency due to bi-allelic mutations in CYP24A1 causes elevated serum vitamin D, hypercalciuria, nephrocalcinosis, and renal stones.

GNE myopathy, previously termed hereditary inclusion body myopathy (HIBM), is an adult-onset neuromuscular disorder characterized by progressive muscle weakness. The disorder results from biallelic mutations in GNE, encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, the key enzyme of sialic acid synthesis. GNE myopathy, associated with impaired glycan sialylation, has no approved therapy. Here we test potential sialylation-increasing monosaccharides for their effectiveness in prophylaxis (at the embryonic and neonatal stages) and therapy (after the onset of symptoms) by evaluating renal and muscle hyposialylation in a knock-in mouse model (Gne p.M712T) of GNE myopathy. We demonstrate that oral mannosamine (ManN), but not sialic acid (Neu5Ac), mannose (Man), galactose (Gal), or glucosamine (GlcN), administered to pregnant female mice has a similar prophylactic effect on renal hyposialylation, pathology and neonatal survival of mutant offspring, as previously shown for N-acetylmannosamine (ManNAc) therapy. ManN may be converted to ManNAc by a direct, yet unknown, pathway, or may act through another mode of action. The other sugars (Man, Gal, GlcN) may either not cross the placental barrier (Neu5Ac) and/or may not be able to directly increase sialylation. Because GNE myopathy patients will likely require treatment in adulthood after onset of symptoms, we also administered ManNAc (1 or 2g/kg/day for 12 weeks), Neu5Ac (2 g/kg/day for 12 weeks), or ManN (2 g/kg/day for 6 weeks) in drinking water to 6 month old mutant Gne p.M712T mice. All three therapies markedly improved the muscle and renal hyposialylation, as evidenced by lectin histochemistry for overall sialylation status and immunoblotting of specific sialoproteins. These preclinical data strongly support further evaluation of oral ManNAc, Neu5Ac and ManN as therapy for GNE myopathy and conceivably for certain glomerular diseases with hyposialylation.

Pathological glomerular hyposialylation has been implicated in certain unexplained glomerulopathies, including minimal change nephrosis, membranous glomerulonephritis, and IgA nephropathy. We studied our previously established mouse model carrying a homozygous mutation in the key enzyme of sialic acid biosynthesis, N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. Mutant mice died before postnatal day 3 (P3) from severe glomerulopathy with podocyte effacement and segmental glomerular basement membrane splitting due to hyposialylation. Administration of the sialic acid precursor N-acetylmannosamine (ManNAc) led to improved sialylation and survival of mutant pups beyond P3. We determined the onset of the glomerulopathy in the embryonic stage. A lectin panel, distinguishing normally sialylated from hyposialylated glycans, used WGA, SNA, PNA, Jacalin, HPA, and VVA, indicating glomerular hyposialylation of predominantly O-linked glycoproteins in mutant mice. The glomerular glycoproteins nephrin and podocalyxin were hyposialylated in this unique murine model. ManNAc treatment appeared to ameliorate the hyposialylation status of mutant mice, indicated by a lectin histochemistry pattern similar to that of wild-type mice, with improved sialylation of both nephrin and podocalyxin, as well as reduced albuminuria compared with untreated mutant mice. These findings suggest application of our lectin panel for categorizing human kidney specimens based on glomerular sialylation status. Moreover, the partial restoration of glomerular architecture in ManNAc-treated mice highlights ManNAc as a potential treatment for humans affected with disorders of glomerular hyposialylation.

The bifunctional enzyme uridine diphosphate (UDP)1-N-acetylglucosamine (GlcNAc) 2- epimerase/N-acetylmannosamine (ManNAc) kinase (GNE), encoded by the GNE gene, catalyzes the first two committed, rate-limiting steps in the biosynthesis of N-acetylneuraminic acid (Neu5Ac) (1, 2). Neu5Ac is the most abundant mammalian sialic acid and the precursor of most naturally existing sialic acids (3). Sialic acids are negatively charged, terminal residues on glycoconjugates, and assist in many cellular functions including cell-cell interactions, proliferation, and viral or bacterial infections (3, 4). The GNE enzyme consists of two enzymatic domains. The N-terminal domain carries out UDP-GlcNAc epimerase function, whereas the Cterminal domain is responsible for ManNAc kinase activity. In mammals, the end product of sialic acid synthesis, CMP-Neu5Ac, feedback-inhibits UDP-GlcNAc 2-epimerase activity of GNE by binding to its allosteric site (5). Two distinct human disorders, sialuria (OMIM 269921) and hereditary inclusion body myopathy (HIBM; OMIM 600737), are associated with predominantly missense mutations in GNE. Sialuria is an autosomal dominant disorder characterized by coarse facies, variable developmental delay, hepatomegaly and recurrent infections. To date, only seven sialuria patients are described worldwide. All patients have a heterozygous missense mutation affecting the allosteric site of GNE, leading to loss of feedback-inhibition of GNE-epimerase activity by CMP-Neu5Ac, resulting in excessive sialic acid production (6, 7). HIBM and its allelic Japanese disorder, distal myopathy with rimmed vacuoles, or DMRV (OMIM 605820), is an autosomal recessive neuromuscular disorder of adult onset, characterized by slowly progressive muscle weakness and atrophy. More than 500 HIBM\DMRV patients exist worldwide, harboring over 60 GNE mutations. HIBM\DMRV patients have recessive (predominantly missense) mutations in either enzymatic domain of GNE, leading to decreased enzyme activity and, presumably, decreased sialic acid production (2, 8, 9). Whether hyposialylation is the main cause of the neuromuscular symptoms in HIBM\DMRV patients remains unknown. In prokaryotes, GNE epimerase and kinase functions are carried out by two separate enzymes, and prokaryotic 2-epimerases have no allosteric feedback inhibition. In mammals, a bifunctional enzyme might have evolved by gene fusion of the two independent enzymes responsible for epimerase/kinase activity. Similarities between mammalian GNE N-terminal regions with prokaryotic UDP-GlcNAc 2-epimerases and mammalian GNE C-terminal regions with members of the sugar kinase superfamily previously assisted in identifying characteristic motifs of the GNE epimerase and kinase enzymatic domains (10, 11). Bacterial 2-epimerases are allosterically regulated by its substrate UDP-GlcNAc. A structural basis for allosteric activation was demonstrated by a crystallographic analysis of the B. subtilis 2 2-epimerase in complex with the reaction intermediate UDP (12). In addition, the crystallographic structures of the E. coli GNE enzyme unbound and in complex with UDP-N-acetylglucosamine (pdb code 1f6d, 1vgv), and the V. cholera (pdb code 1dzc) and B. anthracis (pdb code 3beo) enzymes in complex with UDP-N-acetylglucosamine were solved. Similarity of the N-terminal domain of the H. sapiens GNE to V. cholera (27% homology), E. coli (20% homology) and B. anthracis (18% homology) 2-epimerases was used to model its three-dimensional structure. In previous studies, structural elements and important ATP, ADP, Mg2+ and substrate-binding amino acids were assigned on the basis of these similarities (10, 11). The N-terminal epimerase domain of the human GNE enzyme contains two α/β domains (domains I and II) that form a cleft at the domain interface harboring the active site. Topology of both domains is similar to the Rossmann dinucleotide binding fold (13). Rossmann fold domains are conserved among mammalian and bacterial 2- epimerases. The human N-terminal GNE epimerase domain has a 7-stranded parallel β-sheet sandwiched between a total of 7 α-helices. The C-terminus of the GNE epimerase domain contains a 6-stranded β-sheet surrounded by a total of 7 α-helices (11). Other carriers of the Rossmann fold, including glycosyltransferases and the epimerase domains of 2-epimerases, have similar N-terminal and C-terminal domains (14, 15). The crystallographic structure of the ManNAc kinase domain of human GNE is solved at 2.84 A resolution (pdb code 3eo3) (16). Residues 409–431 of the mammalian GNE ManNAc kinase domain showed similarities with the phosphate 1 motif of the ATP-binding domain of eukaryotic hexokinases (10). Similar to hexokinases (17), mammalian GNE ManNAc kinase contains a 5-stranded β-sheet β3β2β1β4β5 with β2 being anti-parallel to four other parallel strands with a pair of parallel alpha-helices located on each side of the β-sheet (Domain I). Another 5-stranded β-sheet β8β7β6β9β1 with β7 being anti-parallel to four other parallel strands is surrounded by a pair of parallel α-helices on each side (Domain II). The structure of two similar domains involved in ATP binding is a common feature of the ASKHA (Acetate and Sugar Kinase/Hsp70/Actin) superfamily, described in detail for the bacterial poly(P)/ATP-glucomannokinase (18, 19). Recently, different human GNE mRNA splice variants and three predicted translated proteins, hGNE1, hGNE2 and hGNE3 were described (20, 21). Subsequently, two different mouse Gne mRNA splice variants were described, Gne1 and Gne2, together with their expression in selected tissues (22). In the current study we identified additional human isoforms hGNE4-8, and demonstrate expression of hGNE isoform transcripts in a wide variety of tissues. It is unknown which role these isoforms play in GNE regulation, or GNE-related disease pathology. Based on our previous modeling results of the hGNE1 isoform (11), we now analyze and compare the structural features, with respect to catalytic activity, ligand binding and allosteric regulation, of all eight human GNE isoforms.

Costeff Syndrome, which is caused by mutations in the OPTIC ATROPHY 3 (OPA3) gene, is an early-onset syndrome characterized by urinary excretion of 3-methylglutaconic acid (MGC), optic atrophy and movement disorders, including ataxia and extrapyramidal dysfunction. The OPA3 protein is enriched in the inner mitochondrial membrane and has mitochondrial targeting signals, but a requirement for mitochondrial localization has not been demonstrated. We find zebrafish opa3 mRNA to be expressed in the optic nerve and retinal layers, the counterparts of which in humans have high mitochondrial activity. Transcripts of zebrafish opa3 are also expressed in the embryonic brain, inner ear, heart, liver, intestine and swim bladder. We isolated a zebrafish opa3 null allele for which homozygous mutants display increased MGC levels, optic nerve deficits, ataxia and an extrapyramidal movement disorder. This correspondence of metabolic, ophthalmologic and movement abnormalities between humans and zebrafish demonstrates a phylogenetic conservation of OPA3 function. We also find that delivery of exogenous Opa3 can reduce increased MGC levels in opa3 mutants, and this reduction requires the mitochondrial localization signals of Opa3. By manipulating MGC precursor availability, we infer that elevated MGC in opa3 mutants derives from extra-mitochondrial HMG-CoA through a non-canonical pathway. The opa3 mutants have normal mitochondrial oxidative phosphorylation profiles, but are nonetheless sensitive to inhibitors of the electron transport chain, which supports clinical recommendations that individuals with Costeff Syndrome avoid mitochondria-damaging agents. In summary, this paper introduces a faithful Costeff Syndrome model and demonstrates a requirement for mitochondrial OPA3 to limit HMG-CoA-derived MGC and protect the electron transport chain against inhibitory compounds.

A critical role of the FOX transcription factors in the development of different tissues has been shown. Among these genes, FOXN1 encodes a protein whose alteration is responsible for the Nude/SCID phenotype. Recently, our group reported on a human Nude/SCID fetus, which also had severe neural tube defects, namely anencephaly and spina bifida. This led to hypothesize that FOXN1 could have a role in the early stages of central nervous system development. Here we report on a second fetus that carried the R255X homozygous mutation in FOXN1 that has been examined for the presence of CNS developmental anomalies. At 16 postmenstrual weeks of gestation, the abdominal ultrasonography of the Nude/SCID fetus revealed a morphologically normal brain, but with absence of cavum septi pellucidi (CSP). Moreover, after confirmation of the diagnosis of severe Nude/SCID, the fetus was further examined postmortem and a first gross examination revealed an enlargement of the interhemispheric fissure. Subsequently, a magnetic resonance imaging failed to identify the corpus callosum in any section. In conclusion, our observations did not reveal any gross abnormalities in the CNS anatomy of the Nude/SCID fetus, but alteration of the corpus callosum, suggesting that FOXN1 alterations could play a role as a cofactor in CNS development in a similar fashion to other FOX family members.

Dominant disease alleles are attractive therapeutic targets for allele-specific gene silencing by small interfering RNA (siRNA). Sialuria is a dominant disorder caused by missense mutations in the allosteric site of GNE, coding for the rate-limiting enzyme of sialic acid biosynthesis, UDP-GlcNAc 2-epimerase/ManNAc kinase. The resultant loss of feedback inhibition of GNE-epimerase activity by CMP-sialic acid causes excessive production of free sialic acid. For this study we employed synthetic siRNAs specifically targeting the dominant GNE mutation c.797G>A (p.R266Q) in sialuria fibroblasts. We demonstrated successful siRNA-mediated down-regulation of the mutant allele by allele-specific real-time PCR. Importantly, mutant allele-specific silencing resulted in a significant decrease of free sialic acid, to within the normal range. Feedback inhibition of GNE-epimerase activity by CMP-sialic acid recovered after silencing demonstrating specificity of this effect. These findings indicate that allele-specific silencing of a mutated allele is a viable therapeutic strategy for autosomal dominant diseases, including sialuria.—Klootwijk, R. D., Savelkoul, P. J. M., Ciccone, C., Manoli, I., Caplen, N. J., Krasnewich, D. M., Gahl, W. A., Huizing, M. Allele-specific silencing of the dominant disease allele in sialuria by RNA interference.

Mutations in the key enzyme of sialic acid biosynthesis, uridine diphospho-N-acetylglucosamine 2-epimerase/N-acetylmannosamine (ManNAc) kinase (GNE/MNK), result in hereditary inclusion body myopathy (HIBM), an adult-onset, progressive neuromuscular disorder. We created knockin mice harboring the M712T Gne/Mnk mutation. Homozygous mutant (Gne(M712T/M712T)) mice did not survive beyond P3. At P2, significantly decreased Gne-epimerase activity was observed in Gne(M712T/M712T) muscle, but no myopathic features were apparent. Rather, homozygous mutant mice had glomerular hematuria, proteinuria, and podocytopathy. Renal findings included segmental splitting of the glomerular basement membrane, effacement of podocyte foot processes, and reduced sialylation of the major podocyte sialoprotein, podocalyxin. ManNAc administration yielded survival beyond P3 in 43% of the Gne(M712T/M712T) pups. Survivors exhibited improved renal histology, increased sialylation of podocalyxin, and increased Gne/Mnk protein expression and Gne-epimerase activities. These findings establish this Gne(M712T/M712T) knockin mouse as what we believe to be the first genetic model of podocyte injury and segmental glomerular basement membrane splitting due to hyposialylation. The results also support evaluation of ManNAc as a treatment not only for HIBM but also for renal disorders involving proteinuria and hematuria due to podocytopathy and/or segmental splitting of the glomerular basement membrane.

Background Hereditary Inclusion Body Myopathy (HIBM) is an autosomal recessive, adult onset, non-inflammatory neuromuscular disorder with no effective treatment. The causative gene, GNE, codes for UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase, which catalyzes the first two reactions in the synthesis of sialic acid. Reduced sialylation of muscle glycoproteins, such as α-dystroglycan and neural cell adhesion molecule (NCAM), has been reported in HIBM. Methods We treated 4 HIBM patients with intravenous immune globulin (IVIG), in order to provide sialic acid, because IgG contains 8 μmol of sialic acid/g. IVIG was infused as a loading dose of 1 g/kg on two consecutive days followed by 3 doses of 400 mg/kg at weekly intervals. Results For all four patients, mean quadriceps strength improved from 19.0 kg at baseline to 23.2 kg (+22%) directly after IVIG loading to 25.6 kg (+35%) at the end of the study. Mean shoulder strength improved from 4.1 kg at baseline to 5.9 kg (+44%) directly after IVIG loading to 6.0 kg (+46%) at the end of the study. The composite improvement for 8 other muscle groups was 5% after the initial loading and 19% by the end of the study. Esophageal motility and lingual strength improved in the patients with abnormal barium swallows. Objective measures of functional improvement gave variable results, but the patients experienced improvements in daily activities that they considered clinically significant. Immunohistochemical staining and immunoblotting of muscle biopsies for α-dystroglycan and NCAM did not provide consistent evidence for increased sialylation after IVIG treatment. Side effects were limited to transient headaches and vomiting. Conclusion The mild benefits in muscle strength experienced by HIBM patients after IVIG treatment may be related to the provision of sialic acid supplied by IVIG. Other sources of sialic acid are being explored as treatment options for HIBM.

Many species of insects are known to produce and secrete benzoquinone derivatives. These compounds are usually defined as "defense chemicals." However, in many cases, it has not been determined what the chemicals are meant to defend against. Tribolium beetles produce up to three benzoquinones, but their specific function is not known. In this experiment, one of the derivatives, methyl-1,4-benzoquinone (MBQ) was tested for its effectiveness for one of its purported functions as an antimicrobial. Methyl-1,4-benzoquinone was added in three concentrations (0.3, 30, and 150 microg/ml) to the liquid media of three species of Bacillus bacteria and eight species from four genera of yeasts, and the effect on their growth was monitored. The presence of MBQ altered growth in all species. The bacteria responded more negatively than the yeasts. All bacteria species showed reduced growth at all levels of MBQ. Yeasts were more tolerant to the presence of the chemical, and two species, Saccharomyces microellipsoides and Pichia burtonii, actually showed increased growth at the lowest level of MBQ.

Aberrant glycosylation of dystroglycan occurs in certain muscular dystrophies, including hereditary inclusion body myopathy (HIBM). HIBM harbors a widely varying clinical severity and age of onset, which raised the suspicion of the presence of disease modifier genes. We considered the highly polymorphic dystroglycan gene (DAG1) as a feasible candidate modifier gene. DAG1 genomic DNA was sequenced for 32 HIBM patients, mainly of Persian-Jewish descent. Five novel DAG1 single nucleotide polymorphisms (SNPs) were identified, bringing the total number of SNPs to 19. However, no direct correlation between DAG1 SNPs and clinical severity of HIBM could be detected. Several identified SNPs substitute an amino acid and might modulate dystroglycan function or glycosylation status, and deserve further research. These data are valuable for future studies on the role of DAG1 in HIBM and other muscular dystrophies, especially those dystrophies that involve abnormal glycosylation of dystroglycan.

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The secretory Rab27B small GTPase promotes invasive growth, tumourigenicity and metastasis in oestrogen receptor (ER)-positive human breast cancer cells. Coherently, increased Rab27B expression in breast cancer patients is associated with a poor prognosis. In the present study, bio-energetic profiling revealed that oxidative phosphorylation is significantly reduced in ER-positive breast cancer cells engineered to overexpress Rab27B levels as observed in invasive clinical primary breast cancer. Rab27B-induced metabolic reprogramming to aerobic glycolysis was further evidenced by increased extracellular acidification followed by cathepsin B activation and doxorubicin resistance. Transient silencing of Rab27B and stable transfection of Rab27A, and Rab27B mutants in ER-positive breast cancer cells confirmed that this response was Rab27B-specific and dependent upon Rab27B-GTP activation and vesicle membrane attachment through the C-terminal geranylgeranyl group of this small GTPase. Rab27B-driven extracellular acidification is required and is sufficient to induce filopodia-like morphological changes, primarily involved in the process of cancer cell invasion. Our data demonstrate that a Rab27B-dependent switch from oxidative phosphorylation towards aerobic glycolysis in ER-positive breast cancer cells is accompanied by acidification of the tumour environment.